Treatment and diagnosis of inflammatory disorders and hiv

ABSTRACT

This invention concerns compositions and methods of treating or diagnosing inflammatory disorders and other disorders, as well as compositions and methods of treating HIV.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 16/262,314, filed Jan. 30, 2019, now U.S. Pat. No. 10,603,354,issued Mar. 31, 2020, which is a divisional of U.S. patent applicationSer. No. 15/833,544, filed Dec. 6, 2017, now U.S. Pat. No. 10,226,506issued Mar. 12, 2019, which is a divisional of U.S. patent applicationSer. No. 15/080,089, filed Mar. 24, 2016, now U.S. Pat. No. 9,849,156issued Dec. 26, 2017, which is a divisional of U.S. patent applicationSer. No. 14/563,421, filed Dec. 15, 2014, now U.S. Pat. No. 9,295,710issued Mar. 29, 2016, which is a divisional of U.S. patent applicationSer. No. 13/446,949, filed Apr. 13, 2012, now U.S. Pat. No. 8,926,990issued Jan. 6, 2015, which is a continuation-in-part of InternationalP.C.T. Application No. PCT/US10/56864, filed Nov. 16, 2010, which claimspriority under U.S.C. § 119(e) to U.S. Provisional Application Ser. No.61/261,984, filed Nov. 17, 2009. U.S. patent application Ser. No.13/446,949 is also a continuation-in-part of International P.C.T.Application No. PCT/US10/52453, filed Oct. 13, 2010, which claimspriority under U.S.C. § 119(e) to U.S. Provisional Application Ser. No.61/285,378, filed Dec. 10, 2009 and U.S. Provisional Application Ser.No. 61/251,171, filed Oct. 13, 2009. U.S. patent application Ser. No.15/080,089 is also a divisional of U.S. patent application Ser. No.13/446,949, filed Apr. 13, 2012, now U.S. Pat. No. 8,926,990 issued Jan.5, 2015. The entire disclosures of the applications noted above areincorporated herein by reference in their entirety.

GOVERNMENT INTERESTS

The invention disclosed herein was made, at least in part, withGovernment support under Grant No. NIH R01DE16133 and Grant No. NIHR01DE16133 from the National Institutes of Health. Accordingly, the U.S.Government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to reagents and methods for treating ordiagnosing inflammatory disorders and other disorders, as well as toreagents and methods for removing HIV-infected cells from a subject and,thereby, treating HIV infection.

BACKGROUND OF THE INVENTION

Inflammatory disorders are disorders characterized by the abnormalactivation and subsequent migration of white blood cells (WBCs) toaffected areas of the body. These conditions encompass a wide range ofailments that affect the lives of millions of people throughout theworld. Although some treatments are presently available, many possesssignificantly side effects or are not very effective in alleviating allsymptoms. At the same time, few tests exist that reliably diagnose ormonitor the progress of the diseases. Thus, there is a need for drugsand reagents for treatment and diagnosis of inflammatory disorders.

Human immunodeficiency virus (HIV) is a lentivirus that causes acquiredimmunodeficiency syndrome (AIDS), a condition in humans characterized byseveral clinical features including wasting syndromes, central nervoussystem degeneration and profound immunosuppression that results inlife-threatening opportunistic infections and malignancies. Two closelyrelated types of HIV, i.e., HIV-1 and HIV-2, have been identified. Ofthem, HIV-1 is the most common cause of AIDS. Current efforts to treatHIV infection and prevent HIV transmission have focused onantiretroviral therapy (ART) and topical microbicides that kill thevirus at the primary entry points or on vaccines that elicitvirus-neutralizing antibodies. These strategies target the virusdirectly. However, given the tremendous genetic and antigenicvariability and the high mutation rates of HIV-1, designing vaccines anddrugs that are effective against diverse virus isolates circulatingworldwide today has been a formidable challenge. Thus, there is a needfor novel therapeutic agents and methods for treatment or inhibition ofHIV infection.

SUMMARY OF THE INVENTION

This invention relates to treatment and diagnosis of inflammatorydisorders, e.g., autoimmune diseases, using leukotoxin (LtxA), abacterial protein, as well as to compositions and methods to treat,reduce and prevent HIV infection using LtxA. Shown below are thepolypeptide and nucleotide sequences of LtxA.

Aggregatibacter actinomycetemcomitans strain NJ4500 protein sequence(SEQ ID NO: 1) MATTSLLNTKQQAAQFANSVADRAKENIDAAKEQLQKALDKLGKTGKKLTLYIKNYKKGNGLTALIKAAQKLGIEVYHEGKDGPALTNGILNTGKKLLGLTERGLTLFAPELDKWIQGNKHLSNSVGSTGNLTKAIDKVQSVLGTLQAFLNTAFSGMDLDALIKARQNGKNVTDVQLAKASLNLINELIGTISSITNNVDTFSKQLNKLGEALGQVKHFGSFGDKLKNLPKLGNLGKGLGALSGVLSAISAALLLANKDADTATKAAAAAELTNKVLGNIGKAITQYLIAQRAAAGLSTTGPVAGLIASVVSLAISPLSFLGIAKQFDRARMLEEYSKRFKKFGYNGDSLLGQFYKNTGIADAAITTINTVLSAIAAGVGAASAGSLVGAPIGLLVSAITSLISGILDASKQAVFEHIANQLADKIKAWENKYGKNYFENGYDARHSAFLEDSLKLFNELREKYKTENILSITQQGWDQRIGELAGITRNGDRIQSGKAYVDYLKKGEELAKHSDKFTKQILDPIKGNIDLSGIKGSTTLTFLNPLLTAGKEERKTRQSGKYEFITELKVKGRTDWKVKGVPNSNGVYDFSNLIQHAVTRDNKVLEARLIANLGAKDDYVFVGSGSTIVNAGDGYDVVDYSKGRTGALTIDGRNATKAGQYKVERDLSGTQVLQETVSKQETKRGKVTDLLEYRNYKLDYYYTNKGFKAHDELNSVEEIIGSTLRDKFYGSKFNDVFHGHDGDDLIYGYDGDDRLYGDNGNDEIHGGQGNDKLYGGAGNDRLFGEYGNNYLDGGEGDDHLEGGNGSDILRGGSGNDKLFGNQGDDLLDGGEGDDQLAGGEGNDIYVYRKEYGHHTITEHSGDKDKLSLANINLKDVSFERNGNDLLLKTNNRTAVTFKGWFSKPNSSAGLDEYQRKLLEYAPEKDRARLKRQFELQRGKVDKSLNNKVEEIIGKDGERITSQDIDNLFDKSGNKKTISPQELAGLIKNKGKSSSLMSSSRSSSMLTQKSGLSNDISRIISATSGFGSSGKALSASPLQTNNNFNSYANSL ATTAAAggregatibacter actinomycetemcomitans strain NJ4500 DNA sequence(SEQ ID NO: 2) ATGGCAACTACTTCACTGCTAAATACAAAACAGCAAGCTGCACAGTTTGCAAATTCAGTTGCAGATAGAGCTAAGGAAAATATTGATGCTGCAAAAGAACAATTGCAAAAGGCGTTAGATAAATTAGGGAAGACAGGTAAGAAATTAACTTTATATATCCCTAAGAATTACAAAAAAGGAAATGGTCTTACTGCGCTTATAAAAGCAGCACAGAAGTTAGGGATTGAAGTATATCATGAAGGGAAAGACGGCCCGGCATTAACTAATGGTATTTTAAATACTGGGAAAAAATTACTTGGTCTTACCGAACGAGGTTTAACTTTATTTGCTCCGGAATTAGATAAATGGATTCAAGGTAATAAACATTTAAGTAATTCTGTGGGTAGTACTGGAAATTTGACAAAAGCGATAGATAAGGTTCAGAGTGTTCTTGGTACGTTACAAGCGTTTTTGAACACCGCATTTTCGGGCATGGATTTAGATGCCTTAATTAAAGCCCGTCAAAATGGTAAAAATGTAACAGATGTACAGCTAGCAAAAGCCAGTCTTAACCTGATTAATGAATTGATTGGTACTATTTCTAGCATTACAAATAATGTAGATACTTTTTCTAAACAACTTAATAAGTTAGGTGAAGCACTAGGACAAGTAAAACATTTTGGTAGTTTTGGAGATAAATTAAAGAATTTACCTAAGTTAGGTAATCTTGGAAAAGGTTTAGGTGCATTATCCGGTGTATTGTCGGCTATATCAGCGGCTCTATTACTTGCAAATAAAGATGCTGATACTGCAACGAAAGCAGCGGCTGCAGCTGAATTGACAAATAAAGTGCTAGGTAACATCGGTAAAGCGATCACACAATACTTGATTGCTCAACGTGCTGCAGCGGGGcTTTCTACTACGGGACCTGTCGCAGGGTTAATTGCCTCTGTGGTCAGCTTGGCAATCAGCCCTTTGTCTTTCCTAGGTATTGCGAAACAATTTGATCGTGCGAGAATGCTTGAGGAATACTCGAAACGCTTTAAGAAATTTGGTTATAACGGCGATAGTTTACTTGGTCAATTCTACAAAAATACAGGGATCGCAGATGCTGCGATTACAACGATTAACACTGTATTAAGTGCTATTGCAGCAGGGGTTGGTGCAGCCTCCGCCGGTTCTTTAGTTGGTGCGCCAATCGGTTTGTTAGTGAGTGCGATTACCAGCTTAATTTCAGGAATTCTTGATGCTTCTAAACAAGCCGTTTTTGAACATATCGCGAATCAGCTCGCCGATAAAATTAAAGCATGGGAGAATAAGTACGGTAAGAATTACTTTGAAAATGGCTATGATGCCCGTCATTCCGCCTTCTTGGAAGATTCACTAAAATTATTTAATGAGTTACGTGAAAAATATAAAACCGAAAATATATTATCTATCACTCAACAAGGTTGGGATCAGCGCATTGGTGAATTAGCAGGTATCACTCGTAATGGAGATCGTATTCAAAGTGGTAAAGCTTATGTGGATTATTTGAAAAAGGGTGAGGAGCTTGCAAAGCATAGCGATAAATTCACTAAACAGATTTTAGATCCAATCAAAGGTAATATTGATCTTTCGGGTATaAAAGGTTCTACCACTCTAACTTTTTTAAATCCGTTGTTAACCGCAGGTAAGGAAGAACGGAAAACACGTCAGTCAGGTAAATATGAATTTATTACTGAATTAAAAGTAAAAGGACGTACCGATTGGAAGGTAAAAGGTGTTCCTAATTCTAATGGTGTATATGATTTTTCTAACTTAATTCAACATGCCGTTACACGTGATAATAAAGTTCTAGAAGCAAGATTAATTGCTAATTTGGGTGCTAAAGATGATTATGTTTTTGTCGGATCCGGTTCAACAATAGTTAATGCTGGAGACGGTTATGATGTGGTGGACTATAGTAAAGGTCgCACCGGTGCATTAACAATCGACGGTCGTAATGCTACTAAAGCCGGACAATATAAGGTTGAAAGAGATCTTAGCGGTACTCAAGTCTTGCAGGAAACCGTATCAAAGCAAGAAACTAAACGAGGGAAGGTTACCGATCTACTTGAATATCGTAACTATAAATTAGATTACTATTATACGAATAAGGGCTTTAAAGCTCATGATGAATTAAACTCAGTAGAGGAAATTATCGGCAGCACACTACGTGATAAATTTTATGGTTCTAAATTTAATGATGTTTTCCATGGTCACGATGGCGATGATTTGATTTATGGTTATGATGGCGATGATCGTTTGTATGGCGATAATGGGAATGACGAAATTCATGGCGGCCAAGGTAATGATAAGCTCTATGGTGGTGCCGGTAACGATAGGCTCTTTGGTGAATATGGCAACAACTATCTTGACGGTGGAGAAGGCGACGACCACTTAGAGGGAGGCAATGGTTCCGATATTCTAAGAGGTGGAAGTGGCAATGATAAGTTGTTTGGAAACCAAGGAGATGATTTACTTGACGGTGGAGAAGGCGATGACCAACTTGCCGGTGGAGAAGGAAATGATATTTATGTTTACCGTAAAGAATATGGGCACCACACTATTACGGAACATAGCGGTGATAAAGATAAATTATCATTAGCAAATATCAATCTCAAAGATGTGTCATTTGAGCGTAACGGCAATGATCTACTATTGAAAACAAATAATAGAACAGCAGTAACATTTAAAGGATGGTTTAGTAAACCTAATTCATCGGCAGGATTAGATGAGTATCAAAGAAAACTTCTTGAATACGCACCTGAAAAGGATCGTGCACGACTTAAGAGACAATTTGAGTTACAGCGAGGTAAAGTCGACAAATCACTCAATAATAAAGTTGAAGAAATTATCGGTAAAGATGGGGAGCGGATTACTTCGCAAGACATTGATAATCTTTTTGATAAGAGTGGGAACAAAAAGACAATTTCACCTCAAGAGCTTGCCGGACTTATTAAGAATAAAGGTAAGTCAAGTAGCCTTATGTCTTCTTCTCGTTCGTCAAGTATGCTTACACAAAAGTCCGGTTTGTCAAATGATATTAGTCGTATTATTTCAGCAACCAGTGGTTTTGGTTCATCCGGTAAAGCGTTATCCGCTTCGCCATTGCAGACCAATAATAACTTTAACTCTTACGCAAATTCG TTAGCAACTACTGCGGCC

One aspect of this invention feature a method for treating aninflammatory disorder or tuberculosis, comprising administering to asubject in need thereof an effective amount of leukotoxin. Theleukotoxin can be prepared from Aggregatibacter actinomycetemcomitans.In one embodiment, the leukotoxin comprises, consists essentially of, orconsists of the sequence of SEQ ID NO: 1. The inflammatory disorder canbe an autoimmune disease. Examples of the autoimmune disease includepsoriasis, Crohn's disease, ulcerative colitis, rheumatoid arthritis,psoriatic arthritis, multiple sclerosis, lupus, type I diabetes, primarybiliary cirrhosis, an inflammatory bowel disease, and transplantrejection. In a prefer embodiment, the disorder is psoriasis.

A second aspect of this invention features a pharmaceutical composition,comprising a leukotoxin and a pharmaceutical carrier for topicalapplication. The leukotoxin can be prepared from Aggregatibacteractinomycetemcomitans. In one embodiment, the leukotoxin comprises orconsists of the sequence of SEQ ID NO: 1. This composition can be usedin treating inflammatory disorders, in particular, those in the skin.

A third aspect of the invention features a diagnosing method fordetermining whether a subject has an inflammatory disorder, including anautoimmune disease (e.g., psoriasis). The method includes the steps ofobtaining from the subject a test sample that contains white bloodcells; contacting the sample with leukotoxin (e.g., SEQ ID NO: 1); anddetermining the percentage of the white blood cells that bind to theleukotoxin in the sample. The subject is determined to have the disorderif the percentage is at or above a predetermined value. The leukotoxincan be labeled with a detectable agent, such as FITC. The sample can bea blood sample or a biopsy sample. Preferably, the contacting step isconducted at 0-4° C., such as about 0° C. The predetermined value can beone obtained from a control subject that does not have the disorder.

A fourth aspect of the invention features a method for determining theeffectiveness of a treatment in a patient suffering from an inflammatorydisorder, such as an autoimmune disease (e.g., psoriasis). The methodinclude steps of obtaining a sample that contains white blood cells froma patient that has received a treatment; contacting the sample withleukotoxin (e.g., SEQ ID NO: 1); and determining the percentage of thewhite blood cells that bind to the leukotoxin in the sample. Thetreatment is determined to be effective if the percentage is below apredetermined value. Like that in the above-mentioned diagnosing method,the leukotoxin can be labeled with a detectable agent (e.g., FITC); thesample can be a blood sample or a biopsy sample. Also, the contactingstep can be conducted at 0-4° C., such as at about 0° C. Here, thepredetermined value can be a control value obtained from the patientprior to the treatment.

A fifth aspect of this invention features a method to reduce HIVinfected cells in a subject. The method includes identifying a subjecthaving an HIV-infection, and reducing the level of cells expressingactivated LFA-1 using an anti-LFA-1 agent, wherein the anti-LFA-1 agentis a leukotoxin or an antibody. Another aspect of this inventionfeatures a method for inhibiting an infection with HIV in a subject inneed thereof, the method includes identifying a subject at risk of, orsuspected of, having an HIV-infection, and reducing the level of cellsexpressing activated LFA-1 in the subject, using an anti-LFA-1 agent,wherein the anti-LFA-1 agent is a leukotoxin or an antibody, andreducing the level of cells expressing activated LFA-1 in the subject.Another aspect of this invention features a method for treating asubject with an HIV infection comprising administering to a subject inneed thereof a pharmaceutical composition comprising a therapeuticallyeffective amount of an anti-LFA-1 agent, wherein the anti-LFA-1 agent isa leukotoxin or an antibody and reducing the level of cells expressingactivated LFA-1 in the subject. In the aforementioned methods, the cellsinclude HIV-infected cells or the cells are subject to HIV-infection.Examples of such cells include, but not limited to, peripheral bloodmononuclear cells (PBMCs) and CD4+ T cells. The reducing step can beconducted by contacting the cells with an anti-LFA-1 agent, i.e., anagent that specifically binds to and causes death of the cellsexpressing activated LFA-1. Examples of the anti-LFA-1 agent include aleukotoxin or an antibody. The leukotoxin can be prepared fromAggregatibacter actinomycetemcomitans. In one embodiment, the leukotoxincomprises, consists essentially of, or consists of the sequence of SEQID NO: 1. The antibody can be one that specifically binds to activatedLFA-1 and is conjugated with a cytotoxic agent.

The term “cytotoxic agent” refers to a substance that inhibits orprevents the function of cells and/or causes destruction of cells.Examples include a radioisotope, a toxin, a chemotherapeutic agent, adrug, or a growth inhibitory agent. Examples of the toxin include smallmolecule toxins or enzymatically active toxins of bacteria (e.g.,diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcalenterotoxin A), fungi (e.g., α-sarcin, restrictocin), or plant (e.g.,abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin,gelonin, momoridin, trichosanthin, and barley toxin).

In the aforementioned methods, the cells can be contacted with theanti-LFA-1 agent ex vivo or in vivo. In the latter approach, theanti-LFA-1 agent can be administered to the subject in a pharmaceuticalcomposition comprising the anti-LFA-1 agent and a pharmaceuticallyacceptable carrier. The composition can be administered orally,intra-venously, intramuscularly, transdermally, intrarectally, orintravaginally.

Each of the aforementioned methods can further include administering thesubject an anti-HIV agent selected from the group consisting of anucleoside and nucleotide reverse transcriptase inhibitor, anon-nucleoside reverse transcriptase inhibitor, a protease inhibitor, anintegrase inhibitor, an entry inhibitor, and a maturation inhibitor. Inone embodiment, the anti-HIV agent is selected from the group consistingof AZT, dideoxycytidine, dideoxyinosine, raltegravir, maraviroc,bestatin, hCG, levamisole, estrogen, efavirenz, etravirine,indo-methacin, emtricitabine, tenofovir disoproxil fumarate, amprenavir,tipranavir, indinavir, ritonavir, darunavir, enfuvirtide, andgramicidin. Each of the method can also further include a step ofdetermining the level of HIV-infected cells or cells expressingactivated LFA-1 in the subject prior to or after thecontacting/administering step.

A sixth aspect of this invention features a composition, e.g., apharmaceutical composition, that contains the above-mentioned anti-LFA-1agent such as an antibody or LtxA, an anti-HIV agent, and apharmaceutically acceptable carrier. The composition can be used for thetreatment of an HIV infection in a subject or in the manufacture of amedicament for the treatment of HIV infection.

Specific preferred embodiments of the present invention are described inthe following embodiments and claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B is a set of diagrams showing sensitivity ofJurkat-derived cells (FIG. 1A) and PBMCs (FIG. 1B) to LtxA-mediatedcytotoxicity.

FIGS. 2A, 2B and 2C is a set of diagrams showing flow cytometry resultsof healthy human PBMCs' staining with anti-LFA-1 antibody andsensitivity to LtxA.

FIG. 3 is a set of diagrams showing flow cytometry results of cellsstaining with LtxA-FITC.

FIG. 4 is a set of diagrams showing binding of activated PBMCs to brainendothelial cells (HBECs).

FIG. 5 is a diagram showing migration of WBCs across a brain endothelialcell barrier.

FIG. 6 is a set of diagrams showing effects of LtxA and EFALIZUMAB onproliferation of T-cells.

FIG. 7 is a set of diagrams showing effects of LtxA and EFALIZUMAB onproliferation of PBMCs from psoriasis patients.

FIG. 8 is a set of diagrams showing effects of LtxA and EFALIZUMAB onsemiquantitative clinical psoriasis score and epidermal thickness inpsoriasis xenograft transplantation models.

FIG. 9 is a set of diagrams showing effects of LtxA and EFALIZUMAB onpsoriasis pattern scores and parakeratosis scores in the psoriasisxenograft transplantation models.

FIG. 10 is a set of diagrams showing effects of LtxA and EFALIZUMAB onangiogenesis scores and lymphocyte scores in the psoriasis xenografttransplantation models.

FIG. 11 is a diagram showing effects of LtxA and EFALIZUMAB on stratumgranulosum scores in the psoriasis xenograft transplantation models.

FIGS. 12A and 12B are diagrams showing sensitivity to LtxA-mediatedcytotoxicity by THP-1, a malignant human monocyte cell line, and PBMCsfrom four healthy adults (FIG. 12A), and by Jurkat-derived T-cellsexpressing wild type LFA-1, activated LFA-1, or no LFA-1 (FIG. 12B) withresults shown as representative of biological duplicates and verticalbars representing standard deviation.

FIG. 13 is a set of flow cytometry histograms showing that LtxAtreatment depleted LFA-1^(hi+) cells from PBMCs of an HIV uninfecteddonor.

FIG. 14 is a set of flow cytometry histograms showing depletion ofLFA-1^(hi+) HIV⁺ T-cells by LtxA.

FIG. 15 is a diagram showing qRT-PCR detection of intracellular HIV DNA.

FIGS. 16A, 16B, and 16C are photographs and diagrams showing that HIVgp120 interaction with quiescent native CD4 T cells triggers LFA-1activation and supramolecular rearrangement. FIG. 16A is a set ofphotographs for one representative cell to show dynamics of quiescentnative CD4 T cells interaction with HIV gp120 (top and bottom panels)and ICAM-1 (middle and bottom panels) over time. FIG. 16B is a set ofphotographs showing morphology of ICAM-1 contact areas made upon theinteraction of native CD4 T cells with gp120 and ICAM-1. Images ofrepresentative cells and the percentages of cells that form symmetrical(top panel) or asymmetrical (bottom panel) ICAM-1 rings are shown. FIG.16C is a set of diagrams showing percentages of cells makinggp120-positive (left) and ICAM-1-positive (right) contacts out of thetotal number of cells seen in fields, representing the averages+/−SEM ofthree independent experiments.

FIG. 17 is a set of diagrams showing effects on cell viability of theinteraction of quiescent CD4 T cells with: surface-bound wild typegp120, a mutant gp120 lacking CD4-binding site and V3, R5 gp120 (JRFL),X4 gp120 (HXB2), anti-CD3/anti-CD28, and soluble gp120, where data fromone of two or more representative experiments are shown and the averagesand standard deviation from duplicate wells are presented.

FIG. 18 is a set of diagrams showing that CD4 T cells bearing HIV p24antigen express higher levels of surface LFA-1 expression.

FIGS. 19A and 19B are a set of diagrams showing reduction of viral DNAin HIV-infected PBMCs due to LtxA cytotoxicity, where averages andstandard deviation from 4-5 repeat experiments are presented.

FIG. 20 is a set of photographs showing transient interaction betweennative CD4 T cells and ICAM-1 bilayers, where the same representativefield is shown at the indicated time points; black arrows show the cellsthat interacted transiently with the bilayers at 15, 30, and 50 min.

FIGS. 21A and 21B are diagrams showing the background p24 staining ofCD4 T cells (CD3+CD8-) from a HIV-seronegative donor, NG05 (FIG. 21A),and CD3 expression on p24+ and p24− CD4 T cell populations fromHIV-infected subjects PS05 and PS07 (FIG. 21B).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to reagents and methods for treating ordiagnosing inflammatory disorders, such as autoimmune diseases, as wellas to compositions and methods to treat, reduce and prevent HIVinfection using LtxA.

Autoimmune Compositions and Methods

There are more than 80 such diseases characterized by the chronicactivation of immune cells under non-activating conditions. A majorplayer in the etiology of these conditions is leukocyte functionantigen-1 (LFA-1).

LFA-1, a β2-integrin on the surface of white blood cells, is composed ofCD11a and CD18 and involved in immune cell migration and signaling. Inthe absence of infection, circulating WBCs express a “resting state”LFA-1 on their surface. These WBCs play an essential role in immunesurveillance, waiting to be called upon by the immune system. During aninfection, WBCs need to migrate to the site of insult to destroy theinvading pathogens. Extravasation of WBCs into the infected tissue ismediated by signals, such as cytokines, that are released by host cellsat the infection site. Inflammatory cytokines cause LFA-1 to assume anactive conformation, which results in binding of activated LFA-1 tointercellular adhesion molecule-1 (ICAM-1) on the surface of endothelialcells. The interaction between LFA-1 and ICAM-1 results in migration ofWBCs across the endothelial barrier and into the infected tissue. AsLFA-1 is involved in the migration of immune cells to various sites ofinfection, the chronic activation and upregulation of LFA-1 results inthe migration of these cells to various tissues of the body, resultingin inflammation and organ damage.

One of the most prevalent autoimmune diseases is psoriasis. Thisdisease, for which there is no cure, affects 2-3% of the population (7-8million in U.S.; 125 million worldwide). It results from thehyper-activation of immune cells and keratinocytes in the dermis. Theimmune cells involved in psoriatic lesions have an upregulation andactivation of LFA-1. EFALIZUMAB (RAPTIVA), a recombinant monoclonalantibody which binds to the CD11 subunit of LFA-1, was approved forclinical use in patients with this condition but has been withdrawn fromthe market due to increased risk of viral infection of the centralnervous system (progressive multifocal leukoencephalopathy), bacteriasepsis, invasive fungal disease, and other opportunistic infections.

Current treatment for psoriasis includes topical therapy, phototherapy,and systemic administration of steroids and biologics. Many of thetherapeutic options are not highly targeted (e.g., steroids, anthralin)however, and exhibit toxic side effects (e.g., biologics, cyclosporine,methotrexate, retinoids). Indeed, the most effective therapies are alsoconsidered to have the greatest adverse reactions. Furthermore, overtime, psoriasis can become resistant to specific therapies and thesetreatments are periodically changed to prevent both the development ofdrug resistance and the occurrence of adverse reactions. This practiceis known as treatment rotation.

This invention is based, at least in part, on the unexpected discoveriesthat LtxA efficiently and specifically targets and kills WBCs thatexpress the activated conformation of LFA-1 on their surface whilehaving little or no toxic effect on other cells or organs in the body.As disclosed in the examples below, LtxA is highly effective in treatingpsoriasis with minimal toxicity because of its target specificity.

LtxA

LtxA is a ˜115 kDa protein produced by the Gram negative bacteriumAggregatibacter actinomycetemcomitans (Kachlany, S. C. 2010. J Dent Res89:561-570). LtxA specifically kills leukocytes of humans and Old WorldPrimates by forming pores in the membrane and causing apoptosis ornecrosis (Mangan et al., 1991. Infect Immun 59:3267-72). LtxA bindsspecifically to LFA-1 and cells that lack LFA-1 are resistant to itstoxicity (Kachlany, S. C. et al., 2010. Leukemia Research 34:777-85).For example, LtxA is not active against human red blood cells, humanepithelial cells, rat cells, or mouse cells. LtxA also remains active inthe presence of human peripheral blood.

Since LtxA is able to identify and kill white blood cells resulting fromautoimmune disease, it is an ideal agent for both the detection andtreatment of these conditions. For example, blood from a patient can beanalyzed using LtxA-FITC staining. A finding of a large percentage ofactivated WBCs indicates that the patient should undergo LtxA therapy.The effectiveness of the leukotoxin treatments can be monitored byemploying LtxA-FITC reagent that initially diagnosed the disease. As thepatient responds positively to treatment, the number of WBCs withupregulated activated surface LFA-1 should be seen to decrease. Further,because of LtxA's highly specific targeting ability, few side effectsare expected.

LtxA is able to kill many leukemia and lymphoma cell lines andpreclinical studies have shown that it may be an effective targetedtherapy for treating hematological malignancies. LtxA is expected to bemore advantageous than EFALIZUMAB because it displays a significantlygreater selective action by only targeting active LFA-1 and therebymainly destroying activated leukocytes involved in disease. In non-humanprimates, it was found that a single LtxA treatment depleted leukocytecounts for only 12 hours and high doses administered to mice were foundto be non-toxic.

While many LtxA preparations can be used, highly purified LtxA ispreferred. Examples include LtxA polypeptide purified fromAggregatibacter actinomycetemcomitans (SEQ ID NO: 1 shown above) andother variants having substantially the same biological activity as thathaving the sequence of SEQ ID NO: 1. It was discovered thatAggregatibacter actinomycetemcomitans secreted active LtxA into culturesupernatants (Kachlany, S. C., et al. 2000. Infect Immun 68:6094-100)and an efficient method for its purification was described in Kachlany,S. C., et al. 2002. Protein Expr Purif 25:465-71. This method cantherefore be used to prepare isolated or purified LtxA polypeptide. Inone example, a purification procedure of the toxin involves:

-   -   a. inoculating a single colony of Aggregatibacter        actinomycetemcomitans into a fresh broth and growing cultures;    -   b. adding the growing cultures to fresh broth, adding glass        beads and incubating;    -   c. centrifuging the incubated culture, forming a pellet and a        supernatant;    -   d. filtering the supernatant through a membrane to provided a        filtered supernatant;    -   e. mixing (NH₄)₂SO₄ and the filtered supernatant together to        form a mixture;    -   f. centrifuging the mixture to form a mixture pellet;    -   g. resuspending the mixture pellet in buffer to form a protein        resuspension;    -   h. passing the protein resuspension through a column; and    -   i. collecting the protein eluting off the column.        See also PCT/US2006/45258 (WO 2007/062150) and US Application        20090075883 (U.S. Ser. No. 12/154,843). The contents of these        two documents are incorporated herein by reference.

An “isolated polypeptide” refers to a polypeptide that has beenseparated from other proteins, lipids, and nucleic acids with which itis naturally associated. The polypeptide can constitutes at least 10%(i.e., any percentage between 10% and 100%, e.g., 20%, 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purifiedpreparation. Purity can be measured by any appropriate standard method,for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. An isolated polypeptide of theinvention can be purified from a natural source, produced by recombinantDNA techniques, or by chemical methods. A functional equivalent of LtxArefers to a polypeptide derivative of the LtxA polypeptide, e.g., aprotein having one or more point mutations, insertions, deletions,truncations, a fusion protein, or a combination thereof. It retainssubstantially the activity of the LtxA polypeptide, i.e., the ability totarget and kill WBCs that express the activated conformation of LFA-1 ontheir surface while having little or no toxic effect on other cells ororgans in the body. The isolated polypeptide can contain SEQ ID NO: 1 ora functional fragment of SEQ ID NO: 1. In general, the functionalequivalent is at least 75% (e.g., any number between 75% and 100%,inclusive, e.g., 70%, 80%, 85%, 90%, 95%, and 99%) identical to SEQ IDNO: 1.

All of naturally occurring LtxA, genetic engineered LtxA, and chemicallysynthesized LtxA can be used to practice the invention disclosedtherein. LtxA obtained by recombinant DNA technology may have the sameamino acid sequence as naturally a occurring LtxA (SEQ ID NO: 1) or anfunctionally equivalent thereof. The term “LtxA” also covers chemicallymodified LtxA. Examples of chemically modified LtxA include LtxAsubjected to conformational change, addition or deletion of a sugarchain, and LtxA to which a compound such as polyethylene glycol has beenbound. Once purified and tested by standard methods or according to themethod described in the examples below, LtxA can be included inpharmaceutical composition, e.g., a topical composition.

The amino acid composition of the LtxA polypeptide described herein mayvary without disrupting the ability of the polypeptide to target andkill WBCs. For example, it can contain one or more conservative aminoacid substitutions. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in SEQ IDNO: 1 is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, mutations can be introducedrandomly along all or part of SEQ ID NO: 1, such as by saturationmutagenesis, and the resultant mutants can be screened for the abilityto improve skin condition to identify mutants that retain the activityas descried below in the examples.

A LtxA polypeptide as described in this invention can be obtained as anaturally occurring polypeptide or a recombinant polypeptide. To preparea recombinant polypeptide, a nucleic acid encoding it (e.g., SEQ ID NO:2) can be linked to another nucleic acid encoding a fusion partner,e.g., glutathione-s-transferase (GST), 6×-His epitope tag, or M13 Gene 3protein. The resultant fusion nucleic acid expresses in suitable hostcells a fusion protein that can be isolated by methods known in the art.The isolated fusion protein can be further treated, e.g., by enzymaticdigestion, to remove the fusion partner and obtain the recombinantpolypeptide of this invention.

Compositions

Within the scope of this invention is a composition that contains asuitable carrier and one or more of the active agents described above,e.g., LtxA. The composition can be a pharmaceutical composition thatcontains a pharmaceutically acceptable carrier, or a cosmeticcomposition that contains a cosmetically acceptable carrier.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo. The term “pharmaceutically acceptable carrier” refers to any ofthe standard pharmaceutical carriers, such as a phosphate bufferedsaline solution, water, emulsions, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Apharmaceutically acceptable carrier, after administered to or upon asubject, does not cause undesirable physiological effects. The carrierin the pharmaceutical composition must be “acceptable” also in the sensethat it is compatible with the active ingredient and, preferably,capable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active agent.Examples of other carriers include colloidal silicon oxide, magnesiumstearate, cellulose, and sodium lauryl sulfate.

Pharmaceutical compositions for topical administration according to thepresent invention can be formulated as solutions, ointments, creams,suspensions, lotions, powders, pastes, gels, sprays, aerosols, or oils.Alternatively, topical formulations can be in the form of patches ordressings impregnated with active ingredient(s), which can optionallycomprise one or more excipients or diluents. In some preferredembodiments, the topical formulations include a material that wouldenhance absorption or penetration of the active agent(s) through theskin or other affected areas.

A topical composition contains a safe and effective amount of adermatologically acceptable carrier suitable for application to theskin. A “cosmetically acceptable” or “dermatologically-acceptable”composition or component refers a composition or component that issuitable for use in contact with human skin without undue toxicity,incompatibility, instability, allergic response, and the like. Thecarrier enables an active agent (such as LtxA) and optional component tobe delivered to the skin at an appropriate concentration(s). The carriercan thus act as a diluent, dispersant, solvent, or the like to ensurethat the active materials are applied to and distributed evenly over theselected target at an appropriate concentration. The carrier can besolid, semi-solid, or liquid. Preferably, it is in the form of a lotion,a cream, or a gel, in particular one that has a sufficient thickness oryield point to prevent the active materials from sedimenting. Thecarrier can be inert or possess dermatological benefits of its own. Itshould also be physically and chemically compatible with the activecomponents described herein, and should not unduly impair stability,efficacy, or other use benefits associated with the composition.

The topical composition may be a cosmetic or dermatologic product in theform known in the art for topical or transdermal applications, includingsolutions, aerosols, creams, gels, patches, ointment, lotion, or foam.

The cosmetic composition may contain a wide variety of optionalcomponents, provided that such optional components are physically andchemically compatible with the essential components described herein.Examples of such components include those described in, e.g., U.S. Pat.No. 7,405,195; Harry's Cosmeticology, 7th Ed., Harry & Wilkinson (HillPublishers, London 1982); Pharmaceutical Dosage Forms—Disperse Systems;Lieberman, Rieger & Banker, Vols. 1 (1988) & 2 (1989); Marcel Decker,Inc.; The Chemistry and Manufacture of Cosmetics, 2nd. Ed., deNavarre(Van Nostrand 1962-1965); and The Handbook of Cosmetic Science andTechnology, 1st Ed. Knowlton & Pearce (Elsevier 1993).

The topical composition is useful for treating inflammatory disorders inthe skin, such as psoriasis. In addition, it is useful in regulating orimproving skin condition, including regulating visible or tactilewrinkles or discontinuities in skin, e.g., visible or tactile wrinklesor discontinuities in skin texture or color, more especially thoseassociated with skin inflammation, ageing, or other internal factors(e.g., biochemical changes from within the skin) or external factors(e.g., ultraviolet radiation, environmental pollution, wind, heat, lowhumidity, harsh surfactants, and abrasives).

Treatment Methods

The above-described active agents or a composition containing the agentscan be used to treat an inflammatory disorder. Accordingly, theinvention also features methods for treating in a subject aninflammatory disorder, e.g., an autoimmune disease. Autoimmune diseasesare disorders characterized by the chronic activation of immune cellsunder non-activating conditions. Examples include psoriasis,inflammatory bowel diseases (e.g., Crohn's disease and ulcerativecolitis), rheumatoid arthritis, psoriatic arthritis, multiple sclerosis,lupus, type I diabetes, primary biliary cirrhosis, and transplant.

Among the above-listed diseases, psoriasis is the most prevalentautoimmune disease, affecting 2-3% of the population. Psoriasis is adisease that affects the dermis and results from the hyper-activation ofimmune cells and keratinocytes. It has been found that immune cellsinvolved in psoriatic lesions have an upregulation of LFA-1 (de Boer etal., Archives of dermatological research 1994; 286: 304-311 and McGregoret al., Journal of the American Academy of Dermatology 1992: 27:383-388.) In fact, EFALIZUMAB (RAPTIVA) is a monoclonal antibody therapythat is indicated for the treatment of psoriasis. Thus, LtxA andEFALIZUMAB both target the same cell type.

Crohn's disease and ulcerative colitis are diseases of the GI tract andresult from an influx of immune cells into the intestines. Severalstudies have shown that LFA-1 is upregulated in colonic lymphocytesinvolved in the pathogenesis of Crohn's disease and ulcerative colitis(Bernstein et al., Clinical immunology, Orlando, Fla. 2002; 104: 67-72and Vainer et al., The American journal of surgical pathology 2000; 24:1115-1124).

Multiple sclerosis (MS) is characterized by the influx of immune cellsinto the central nervous system. Studies have also shown upregulatedexpression of LFA-1 on immune cells collected from blood and cerebralspinal fluid from MS patients (Elovaara et al. Neurology 1998; 51:1703-1708; and Elovaara et al., Archives of neurology 2000; 57:546-551). As described below in the example section, LtxA can suppresssymptoms of MS via interfering with cell adhesion and depletehighly-activated immune cells in the CNS.

Rheumatoid arthritis is a disease affecting the joints and occurs whenactivated immune cells migrate to the synovium Immune cells found insynovial fluid from RA patients have enhanced expression of LFA-1 andblocking LFA-1 has proven an effective therapeutic strategy inexperimental animal systems (see, e.g., Singh et al., J Immunol 2008;180: 1971-1978). Thus, LtxA would preferentially affect and eliminatethe immune cells involved in RA pathogenesis.

Systemic lupus erythematosus or lupus is an autoimmune disease thataffects primarily women and for which there is little effectivetreatment. The disease is characterized by immune complex-mediatedtissue injury. Activated immune cells are responsible for the productionof immune complexes. These immune cells express LFA-1 at high levels andseveral studies have shown the critical importance of LFA-1-expressingcells in the development of lupus (see, e.g., Kevil et al., The Americanjournal of pathology; 2004; 165: 609-616). Accordingly, LtxA can be usedin treating systemic lupus erythematosus.

Other examples of inflammatory disorders that can be treated with LtxAinclude asthma, myocardial infarction, stroke, inflammatory dermatoses(e.g., dermatitis, eczema, atopic dermatitis, allergic contactdermatitis, urticaria, necrotizing vasculitis, cutaneous vasculitis,hypersensitivity vasculitis, eosinophilic myositis, polymyositis,dermatomyositis, and eosinophilic fasciitis), acute respiratory distresssyndrome, fulminant hepatitis, hypersensitivity lung diseases (e.g.,hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-typehypersensitivity, interstitial lung disease or ILD, idiopathic pulmonaryfibrosis, and ILD associated with rheumatoid arthritis), and allergicrhinitis. Additional examples also include myasthenia gravis, juvenileonset diabetes, glomerulonephritis, autoimmune throiditis, ankylosingspondylitis, systemic sclerosis, acute and chronic inflammatory diseases(e.g., systemic anaphylaxia or hypersensitivity responses, drugallergies, insect sting allergies, allograft rejection, andgraft-versus-host disease), and Sjogren's syndrome.

In addition to the above-listed inflammation-related disorders, LtxA canalso be used to treat tuberculosis (TB). Approximately one-third of theworld's population is infected with the bacterial agent that causes TB,Mycobacterium tuberculosis. Upon infection, M. tuberculosis enters intoa latent state where it lives within macrophages. Inside the macrophage,the bacterium remains hidden from the immune system and is verydifficult to eradicate. Indeed, bacterial latency and subsequentreactivation are key components of tuberculosis pathogenesis. Aggressiveantibiotic therapy consisting of isoniazid and rifampin is prescribedfor 9-12 months. Because of this prolonged course of therapy, complianceis a major concern and few patients complete the full regimen. It wasreported that M. tuberculosis-infected macrophages have anover-abundance of LFA-1 proteins on their surfaces (DesJardin et al.Microbiology 2002; 148: 3161-3171). This induction of LFA-1 expressionoccurs upon interaction between the bacterium and macrophage host cell.Thus, M. tuberculosis-infected macrophages would also be targets forLtxA and LtxA can be used for depleting latently-infected cells. Thisaction would essentially expose bacteria, thereby eliminating theprotection of the host cell and rendering the pathogen susceptible tothe immune system and systemic antibiotic therapy.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human primates (particularly higher primates), dog, rodent (e.g.,mouse or rat), guinea pig, cat, and non-mammals, such as birds,amphibians, reptiles, etc. In a preferred embodiment, the subject is ahuman. In another embodiment, the subject is an experimental animal oranimal suitable as a disease model.

A subject to be treated for an inflammatory disorder can be identifiedby standard diagnosing techniques for the disorder. Optionally, thesubject can be examined for the level or percentage of WBCs that bind toLtxA in a test sample obtained from the subject by methods describedbelow. If the binding level or percentage is at or above a thresholdvalue (which can be obtained from a normal subject), the subject is acandidate for treatment with an effective amount of LtxA.

“Treating” or “treatment” refers to administration of a compound oragent to a subject, who has a disorder (such as an inflammatorydisorder), with the purpose to cure, alleviate, relieve, remedy, delaythe onset of, or ameliorate the disorder, the symptom of the disorder,the disease state secondary to the disorder, or the predispositiontoward the disorder. A “therapeutically effective amount” refers to theamount of an agent sufficient to effect beneficial or desired results. Atherapeutically effective amount can be administered in one or moreadministrations, applications or dosages and is not intended to belimited to a particular formulation or administration route.

The agent can be administered in vivo or ex vivo, alone orco-administered in conjunction with other drugs or therapy. As usedherein, the term “co-administration” or “co-administered” refers to theadministration of at least two agent(s) or therapies to a subject. Insome embodiments, the co-administration of two or more agents/therapiesis concurrent. In other embodiments, a first agent/therapy isadministered prior to a second agent/therapy. Those of skill in the artunderstand that the formulations and/or routes of administration of thevarious agents/therapies used may vary.

In an in vivo approach, LtxA is administered to a subject. Generally,LtxA is suspended in a pharmaceutically-acceptable carrier (e.g.,physiological saline) and administered orally or by intravenousinfusion, or injected or implanted subcutaneously, intramuscularly,intrathecally, intraperitoneally, intrarectally, intravaginally,intranasally, intragastrically, intratracheally, or intrapulmonarily.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100 mg/kg.Variations in the needed dosage are to be expected in view of thevariety of compounds available and the different efficiencies of variousroutes of administration. Variations in these dosage levels can beadjusted using standard empirical routines for optimization as is wellunderstood in the art. Encapsulation of the compound in a suitabledelivery vehicle (e.g., polymeric microparticles or implantable devices)may increase the efficiency of delivery.

Diagnostic and Prognostic Methods

As discussed above, LFA-1, present on WBCs of inflammatory diseasepatients, can act as a marker to detect and monitor the treatment ofthese afflictions while providing a therapeutic target forpharmaceutical agents. LtxA specifically targets WBCs that express theactivated conformation of LFA-1, and therefore can be used in diagnosingdiseases meditated by such WBCs.

To that end, this invention also features diagnosis methods. WBCsexpressing the activated conformation of LFA-1 can be detected in asubject based on the presence of the binding of LtxA in a test samplefrom the subject. In other words, the binding of LtxA can be used asmarkers to indicate the presence or absence of WBCs involved ininflammatory disorders, including autoimmune diseases. Diagnostic andprognostic assays of the invention include methods for assessing thebinding level of LtxA with WBCs.

The binding level in a test sample can be evaluated by obtaining a testsample from a test subject and contacting the test sample with LtxA. The“test sample” includes tissues, cells and biological fluids isolatedfrom a subject, as well as tissues, cells and fluids present within asubject. The level of binding of LtxA to WBCs can be measured in anumber of ways, including that described in the examples below. In apreferred embodiment, LtxA or its fragments that mediate binding betweenLtxA and LFA-1 (i.e., probes) are labeled with a detectable agent. Theterm “labeled” is intended to encompass direct labeling of the probe byphysically linking a detectable substance to the probe, as well asindirect labeling of the probe by reactivity with a detectablesubstance. For example, LtxA (or its fragment) can be indirectly labeledusing a second antibody directed against LtxA, wherein the secondantibody is coupled to a detectable substance. Examples of detectablesubstances or labels include radio isotopes (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H,or ³²P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase,luciferase, or β-glactosidase), fluorescent moieties or proteins (e.g.,fluorescein isothiocyanate, rhodamine, phycoerythrin, GFP, or BFP), orluminescent moieties (e.g., QDOT™ nanoparticles by the Quantum DotCorporation, Palo Alto, Calif.).

LtxA not only binds to, but also kills, WBCs. In the diagnostic orprognostic method, to minimize any potential errors caused by celldeath, the binding of LtxA and WBCs can be conducted at low temperatures(e.g., 0-4° C.) and for a short period of time such as 5 to 20 or 30minutes.

The prognostic assays described herein can be used to determine whethera subject is suitable to be administered with an agent (e.g., a drug) totreat an inflammatory disorder. For example, such assays can be used todetermine whether a subject can be administered with a cytotoxic drug orimmune-suppressants to treat an autoimmune disorder, including thoseinvolved in organ/tissue transplantation.

Thus, also featured in this invention is a method of monitoring atreatment for inflammatory disorders (e.g., an autoimmune disorder) in asubject. For this purpose, the binding level between LtxA and WBCs canbe determined for test samples from a subject before, during, or afterundergoing a treatment. A decrease in the binding level after thetreatment indicates that the subject can be further treated by the sametreatment. For example, a patient who has received organ or tissuetransplantation often faces the problems of organ or tissue rejection.That is, the body has an immune response to an organ or tissue whichcauses failure of the transplant. To address this problem, organ ortissue transplantation is often accompanied by nonspecific immunesuppression therapy to prevent T cell-mediated rejection. However, theseimmunosuppressants can cause infection, hypertension, cancer, and otherundesirable side effects. Therefore, there is a need for monitoring thesuppression. To that end, LtxA's biding level can serve as a marker fora proper level or degree of immune suppression. A skilled in the art canadjust the amount of immunosuppressants and length of treatment based onthe level of the binding during the course of the treatment.

Information obtained from practice of the above assays is useful inprognostication, identifying progression of, and clinical management ofdiseases and other deleterious conditions affecting an individual'shealth status. The information more specifically assists the clinicianin designing therapy or other treatment regimes to treat inflammatorydisorders, including autoimmune diseases.

HIV Compositions and Methods

HIV infection is characterized by a latent period in which the virusstably integrates into the host genome. In this way, HIV is able to hidefrom the host immune system and lays dormant for potentially years.Reactivation of the virus can occur and result in AIDS.

HIV preferentially infects memory CD4+ T cells, which over-express theadhesion integrin, leukocyte function antigen-1 (LFA-1). LFA-1 is aβ2-integrin on the surface of white blood cells (WBCs). It is composedof CD11a and CD18 and involved in immune cell migration and signaling.In the absence of infection, circulating WBCs express a “resting state”LFA-1 on their surface. LFA-1 and its ICAM-1 ligand play an importantrole in promoting HIV-1 infectivity and transmission. These moleculesare present on the envelope of HIV-1 virions and are integral componentsof the HIV virological synapse. However, cellular activation is requiredto convert LFA-1 to the active conformation that has high affinitybinding for ICAM-1.

As disclosed herein, it was unexpected that HIV-1 gp120 was sufficientto trigger LFA-1 activation in fully quiescent native CD4 T cells in aCD4 and chemokine receptor-dependent manner, and that these CD4 T cellsbecame more susceptible to killing by cytotoxic agents that targetactivated LFA-1. Accordingly, this invention provides cytotoxic agentsthat target activated LFA-1 for eliminating HIV-infected cells, therebytreating or controlling HIV infection.

One example of the LFA-1-targeting cytotoxic agents is LtxA or itsfunctional equivalent. Replicating and residing inside macrophages andT-lymphocytes, HIV viruses are difficult to combat using conventionalanti-retroviral therapy because the viruses “hide” from the immunesystem inside host cells. As disclosed herein, LtxA is highly effectivein killing HIV-infected cells. For example, virus p24-expressing CD4 Tcells in the peripheral blood of HIV-infected subjects were found tohave higher levels of surface LFA-1, and LtxA treatment led tosignificant reduction of the viral DNA burden. These results demonstratethe use of leukotoxin to destroy HIV-1-infected cells independent of thevirus variability and deplete the virus reservoir that cannot beeradicated by the conventional therapy.

Once these infected cells are destroyed, the virus would be released andacted upon by the natural host immune defenses. Otherwise, integratedvirus-containing T-cells would be killed, thus removing the potentialthreat of reactivation at a later time. Leukotoxin treatment in thiscase is different from other therapies in that the therapy is notdirected against the virus (which would select for resistant HIVmutants), but rather against the host cell in which the virus resides.That is, by targeting the cells and specifically the invariable cellularadhesion molecule LFA-1, the aforementioned LtxA treatment circumventsthe problems of virus variability and drug resistance that conventionaltherapy faces.

While many LtxA preparations can be used to practice this invention,highly purified LtxA is preferred. Examples include LtxA polypeptidepurified from Aggregatibacter actinomycetemcomitans (SEQ ID NO: 1 shownabove) and other variants having substantially the same biologicalactivity as that having the sequence of SEQ ID NO: 1. It was discoveredthat Aggregatibacter actinomycetemcomitans secreted active LtxA intoculture supernatants (Kachlany, S. C., et al. 2000. Infect Immun68:6094-100) and an efficient method for its purification was describedin Kachlany, S. C., et al. 2002. Protein Expr Purif 25:465-71. Thismethod can therefore be used to prepare isolated or purified LtxApolypeptide. In one example, a purification procedure of the toxininvolves: (a) inoculating a single colony of Aggregatibacteractinomycetemcomitans into a fresh broth and growing cultures; (b)adding the growing cultures to fresh broth, adding glass beads andincubating; (c) centrifuging the incubated culture, forming a pellet anda supernatant; (d) filtering the supernatant through a membrane toprovided a filtered supernatant; (e) mixing (NH₄)₂SO₄ and the filteredsupernatant together to form a mixture; (f) centrifuging the mixture toform a mixture pellet; (g) resuspending the mixture pellet in buffer toform a protein resuspension; (h) passing the protein resuspensionthrough a column; and (i) collecting the protein eluting off the column.See also PCT/US2006/45258 (WO 2007/062150), US Application 20080318252(U.S. Ser. No. 12/150,038), and US Application 20090075883 (U.S. Ser.No. 12/154,843). The contents of these patent documents are incorporatedherein by reference.

An “isolated polypeptide” refers to a polypeptide that has beenseparated from other proteins, lipids, and nucleic acids with which itis naturally associated. The polypeptide can constitute at least 10%(i.e., any percentage between 10% and 100% inclusive, e.g., 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 99%) by dry weight of thepurified preparation. Purity can be measured by any appropriate standardmethod, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis. An isolated polypeptide of theinvention can be purified from a natural source, produced by recombinantDNA techniques, or by chemical methods. A functional equivalent of LtxArefers to a polypeptide derivative of the LtxA polypeptide, e.g., aprotein having one or more point mutations, insertions, deletions,truncations, a fusion protein, or a combination thereof. It retainssubstantially the activity of the LtxA polypeptide, i.e., the ability totarget and kill cells that express the activated conformation of LFA-1on their surface while having little or no toxic effect on other cellsor organs in the body. The isolated polypeptide can contain SEQ ID NO: 1or a functional fragment of SEQ ID NO: 1. In general, the functionalequivalent is at least 75% (e.g., any number between 75% and 100%,inclusive, e.g., 70%, 80%, 85%, 90%, 95%, and 99%) identical to SEQ IDNO: 1.

All of naturally occurring LtxA, genetic engineered LtxA, and chemicallysynthesized LtxA can be used to practice the invention disclosedtherein. LtxA obtained by recombinant DNA technology may have the sameamino acid sequence as naturally a occurring LtxA (SEQ ID NO: 1) or anfunctionally equivalent thereof. The term leukotoxin or LtxA also coverschemically modified leukotoxin or LtxA. Examples of chemically modifiedLtxA include LtxA subjected to conformational change, addition ordeletion of a sugar chain, and LtxA to which a compound such aspolyethylene glycol has been bound. Once purified and tested by standardmethods or according to the method described in the examples below, LtxAcan be included in pharmaceutical composition, e.g., a topicalcomposition.

The amino acid composition of the LtxA polypeptide described herein mayvary without disrupting the ability of the polypeptide to target andkill WBCs. For example, it can contain one or more conservative aminoacid substitutions. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in SEQ IDNO: 1 is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, mutations can be introducedrandomly along all or part of SEQ ID NO: 1, such as by saturationmutagenesis, and the resultant mutants can be screened for the abilityto kill LFA-1 positive cells to identify mutants that retain theactivity as descried below in the examples.

A LtxA polypeptide as described in this invention can be obtained as anaturally occurring polypeptide or a recombinant polypeptide. Inaddition to leukotoxin or LtxA, other examples of the aforementionedLFA-1-targeting cytotoxic agents include antibody-based cytotoxicagents. The term “antibody” refers to an immunoglobulin molecule orimmunologically active portion thereof, i.e., an antigen-bindingportion. Examples include a protein having at least one, and preferablytwo, heavy (H) chain variable regions (V_(H)), and at least one andpreferably two light (L) chain variable regions (V_(L)). The V_(H) andV_(L) regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (FR). As used herein, the term “immunoglobulin” refers to aprotein consisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognized human immunoglobulin genes includethe kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3,IgG4), delta, epsilon and mu constant region genes, as well as themyriad immunoglobulin variable region genes.

Antibodies that are specifically binds to activated LFA-1 can be madeusing methods known in the art. An anti-LFA-1 antibody can be apolyclonal or a monoclonal antibody. Examples of such antibodies includemonoclonal antibody MEM-83 as marketed by ABCAM PLC (Cambridge, Mass.)and described in Hogg et al. 1993. Am Rev Respir Dis, 148, S55-59, andsimilar antibodies descried in Porter et al. 2002. J Immunol, 168,6330-6335 (e.g., mAb38 and mAb G25.2).

In one embodiment, the antibody can be recombinantly produced, e.g.,produced by phage display or by combinatorial methods. In another neembodiment, the antibody is a fully human antibody (e.g., an antibodymade in a mouse which has been genetically engineered to produce anantibody from a human immunoglobulin sequence), or a non-human antibody,e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camelantibody.

To make antibody based cytotoxic agents, the above-mentioned antibodycan be conjugated to a cytotoxic agent with methods known in the art.Examples of cytotoxic agents include radioactive isotopes (e.g.,phosphorus-32, copper-67, arsenic-77, rhodium-105, palladium-109,silver-111, tin-121, iodine-125 or 131, holmium-166, lutetium-177,rhenium-186 or 188, iridium-194, gold-199, astatium-211, yttrium-90,samarium-153, or bismuth-212), chemotherapeutic agents, e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chloramucil,daunorubicin, or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial (e.g.,Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcalenterotoxin A), fungal (e.g., α-sarcin, restrictocin), plant (e.g.,abrin, ricin, modeccin, viscumin, pokeweed anti-viral protein, saporin,gelonin, momoridin, trichosanthin, barley toxin) or animal origin, e.g.,cytotoxic RNases, such as extracellular pancreatic RNases; DNase I,including fragments and/or variants thereof.

The above-described active agents target an invariant cellular proteinLFA-1. Thus, the activity of the active agents is not affected by thetremendous genetic, biologic, and antigenic variation of HIV-1. Hence,an approach based on these agents to eradicate HIV-infected cellsdiffers from strategies of utilizing toxin-conjugated CD4 or antibodiesthat target productively infected cells via the virus envelope antigenswhich are not only highly variable but are also occluded to variousdegrees in the vast majority of HIV-1 clinical isolates (Kennedy, et al.2006. J Leukoc Biol 80:1175-1182, and Pincus, et al. 2003. AIDS Res HumRetroviruses 19:901-908). The approach disclosed herein is alsodifferent from utilizing LFA-1 antagonists that are aimed to reduce theefficiency of virus-cell interaction for suppressing virus infection andtransmission.

By destroying virus-infected cells that are left unaffected by thecurrent anti-retroviral therapy (ART), the above-described active agents(e.g., LtxA) can work in synergy with ART to significantly lower oreliminate the overall virus burden in HIV-infected individuals. It wasknown that ART can successfully reduce viremia to <50 copies/ml, but thevirus often still replicates at low levels in infected CD4 T cells andother cell types (Brennan, et al. 2009. J Virol 83:8470-8481 andFischer, et al. 2000. AIDS Res Hum Retroviruses 16:1135-1140). Theabove-described active agents (e.g., LtxA) can be used to deplete theseproductively infected cells and eradicate the residual active virusinfection. In addition, intermittent transient viremia is frequentlydetected in well suppressed HIV-infected subjects on ART (Di Mascio etal. 2004. J Virol 78:10566-10573; Fischer, et al. 2000. AIDS Res HumRetroviruses 16:1135-1140; and Nettles et al., 2005. Jama 293:817-829.)Such viral blips are thought to result from activation oflatently-infected cells by antigen recognition or bystanders in a localinflammatory response (Jones et al. 2007. J Acquir Immune Defic Syndr45:483-493). The aforementioned active agents are effective againstlatent infection as well.

Within the scope of this invention is a composition that contains asuitable carrier and one or more of the active agents described above,e.g., LtxA or an anti-LFA-1 antibody based cytotoxic agent. Thecomposition can be a pharmaceutical composition that contains apharmaceutically acceptable carrier.

Pharmaceutically effective compositions of this invention may beadministered to humans and other animals by a variety of methods thatmay include continuous or intermittent administration. Examples ofmethods of administration may include, but are not limited to, oral,rectal, parenteral, intracisternal, intrasternal, intravaginal,intraperitoneal, topical, transdermal, buccal, or as an oral or nasalspray. Accordingly, the pharmaceutically effective compositions may alsoinclude pharmaceutically acceptable additives, carriers or excipients.Such pharmaceutical compositions may also include the active ingredientsformulated together with one or more non-toxic, pharmaceuticallyacceptable carriers specially formulated for oral administration insolid or liquid form, for parenteral injection or for rectaladministration according to standard methods known in the art.

The term “parenteral” administration refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal,intracisternal, intrasternal, subcutaneous and intraarticular injectionand infusion. Injectable mixtures are known in the art and comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions as well as sterile powders forreconstitution into sterile injectable solutions or dispersions justprior to use. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol and the like), vegetableoils (such as olive oil), injectable organic esters (such as ethyloleate) and suitable mixtures thereof.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid and the like. It may also be desirableto include isotonic agents such as sugars, sodium chloride and the like.Prolonged absorption of the injectable pharmaceutical form may bebrought about by the inclusion of agents which delay absorption such asaluminum monostearate and gelatin. Injectable formulations can besterilized, for example, by filtration through a bacterial-retainingfilter or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved or dispersed in sterile wateror other sterile injectable medium just prior to use.

In some cases, to prolong the effect of the drug, it is desirable toslow drug absorption from subcutaneous or intramuscular injection. Thismay be accomplished by using a liquid suspension of crystalline oramorphous material with poor water solubility. The rate of absorption ofthe drug then depends upon its rate of dissolution which, in turn, maydepend upon crystal size and crystalline form. Alternatively, absorptionof a parenterally administered drug form may be delayed by dissolving orsuspending the drug in an oil vehicle.

To prepare the pharmaceutical compositions of the present invention, aneffective amount of the aforementioned agent(s) can be intimatelyadmixed with a pharmaceutically acceptable carrier according toconventional pharmaceutical compounding techniques to produce a dose. Acarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain amounts ofthe active agents which are effective to achieve the desired therapeuticresponse for a particular patient, compositions and mode ofadministration. The selected dosage level will depend upon the activityof the active agents, the route of administration, the severity of thecondition being treated and the condition and prior medical history ofthe patient being treated. However, it is within the skill of the art tostart doses of the agents at levels lower than required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved.

Compositions according to the present invention may also be administeredin combination with other agents to enhance the biological activity ofsuch agents. Such agents may include any one or more of the standardanti-HIV agents which are known in the art, including, but not limitedto, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine(ddI). Additional agents which have shown anti-HIV effects and may becombined with compositions in accordance to the invention include, forexample, raltegravir, maraviroc, bestatin, human chorionic gonadotropin(hCG), levamisole, estrogen, efavirenz, etravirine, indomethacin,emtricitabine, tenofovir disoproxil fumarate, amprenavir, tipranavir,indinavir, ritonavir, darunavir, enfuvirtide, and gramicidin.

The above-described active agents or a composition containing the agentscan be used to treat or inhibit an HIV infection. Accordingly, theinvention also features methods for treating in a subject has, or issuspected of having, an HIV infection.

A subject to be treated can be identified by standard diagnosingtechniques for the disorder. “Treating” or “treatment” also refers toadministration of a compound or agent to a subject, who has a disorder(such as an HIV infection), with the purpose to cure, alleviate,relieve, remedy, delay the onset of, or ameliorate the disorder, thesymptom of the disorder, the disease state secondary to the disorder, orthe predisposition toward the disorder. A therapeutically effectiveamount can be administered in one or more administrations, applicationsor dosages and is not intended to be limited to a particular formulationor administration route.

The agent can be administered in vivo or ex vivo, alone orco-administered in conjunction with other drugs or therapy. In someembodiments, the co-administration of two or more agents/therapies isconcurrent. In other embodiments, a first agent/therapy is administeredprior to a second agent/therapy. Those of skill in the art understandthat the formulations and/or routes of administration of the variousagents/therapies used may vary.

In an in vivo approach, the above-described agent, e.g., LtxA, isadministered to a subject. Generally, LtxA is suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally or by intravenous infusion, or injected or implantedsubcutaneously, intramuscularly, intrathecally, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily. In an ex vivo approach, asubject's blood can be withdrawn and treated with the above-mentionedagent to remove cells expressing activated LFA-1 before the bloodthus-treated is given back to the subject.

The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.01-100 mg/kg.Variations in the needed dosage are to be expected in view of thevariety of agents available and the different efficiencies of variousroutes of administration. Variations in these dosage levels can beadjusted using standard empirical routines for optimization as is wellunderstood in the art. Encapsulation of the agent in a suitable deliveryvehicle (e.g., polymeric microparticles or implantable devices) mayincrease the efficiency of delivery.

Unlike microbicides and ART, which require constant adherence, as aprophylaxis LtxA can be administered once or a few times in a shortcourse, soon after virus exposure or during the early phases of theinfection, in order to purge a substantial fraction, if not all, ofvirus-harboring cells from the infected individuals. A significantreduction of viral burden in HIV-infected individuals should have asignificant impact in preventing or delaying disease progression ofthese individuals, as well as reducing virus transmission to thecommunity. LtxA may also be applied as a therapeutic agent, inconjunction with or after successful ART to eradicate most, if not all,virus-infected cells that remain. Hence, the use of LtxA has thepotential to shorten, or perhaps eliminate, ART, which is currentlyconsidered to be lifelong. A limited course of LtxA treatment will alsoreduce the potential of eliciting the host immune response that mayneutralize this bacterial toxin.

In one example, LtxA may be administered once or a few times in a shortcourse after ART in the acute or chronic phases of the infection.Indeed, three doses of LtxA to leukemic mice were sufficient toeliminate the malignancy and result in long-term disease-freeprogression (Kachlany, S. C. et al., 2010. Leukemia Research 34:777-85).The short-course intermittent administration of LtxA should reduce therisk of severe immunosuppression as reported with the clinical use ofEFALIZUMAB, a monoclonal antibody against the CD11a subunit of LFA-1 fortreatment of psoriasis Korman et al. 2009. Arch Dermatol 145:937-94. Thecapacity of LtxA to purge a substantial fraction of virus-harboringcells from the infected individuals should have a considerable impact indelaying disease progression and decreasing the duration of ART in theseindividuals, as well as reducing virus transmission to the community.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1

In this example, LtxA's targeting of activated LFA-1 on WBCs wasdemonstrated in a study using a T-lymphocyte cell line (Jurkat) thatexpresses a high level of constitutively activated LFA-1 and controlisogenic cells that either express the wild type form of the receptor orlack LFA-1 expression entirely. It was found that the T-lymphocytes wereten times more sensitive to LtxA-mediated toxicity than the controlcells which were not affected.

Malignant human monocyte cell line, THP-1, and peripheral bloodmononuclear cells (PBMCs) PBMCs from four healthy adults were alsotreated with LtxA at different concentrations for 24 hours. Cellviability was then determined by measurement of cellular ATP. Theresults are shown in FIG. 1B. Untreated samples represent a relativeviability of 1.0. The curve for PBMCs represents the average of the fourhuman PBMC samples performed in quadruplicate. The vertical barsrepresent standard deviation. Results shown are representative ofbiological duplicates. As shown in FIG. 1B, LtxA had little effect onnormal, resting human WBCs by examining its effect on normal PBMCs fromhealthy donors. The majority of cells were found to be resistant to LtxAand a drop in viability was only observed at very high drugconcentrations. PBMCs prepared from healthy adults were also stainedwith anti-LFA-1 (CD11a) antibodies and analyzed by flow cytometry. Shownin FIG. 2 are results of cell size (forward scatter) vs. CD11aexpression after 24-hour treatment with a control (“−LtxA”) and LtxA(“LtxA”). As shown in the figures, only the activated cells with highlevels of LFA-1 were affected by LtxA.

To determine whether LtxA can detect cells that had activated LFA-1 ontheir surface, purified LtxA was covalently labeled with a fluorescentsubstituent, fluorescein isothiocyanate (FITC). This allowed the LtxA tobe detected and quantified by fluorescence spectroscopy when used incell binding experiments. Laboratory studies showed that the LtxA-FITCretained full targeting and biological activity indicating that the FITCmodification did not adversely affect toxin structure. Test cells weremixed with LtxA-FITC for 30 minutes at 0° C. (on ice) and the resultingconjugates were analyzed by flow cytometry.

The test cells included K562 cells (which do not express LFA-1 and areunaffected by LtxA), THP-1 cells, HL-60 cells, and PBMCs (from a healthydonor). As shown in FIG. 3, LtxA-FITC did not kill the test cells due tothe low temperatures and brief time period used. Further observationsdetermined that K562 cells (which lack CD11a and CD18) did not bind theleukotoxin-FITC, indicating that LFA-1 is required for cell staining. Incontrast, LtxA-FITC strongly attached itself to THP-1 cells and withslightly less intensity to HL-60 cells. Similar experiments were carriedout with PBMCs, which possess minimal levels of activated surface LFA-1.As expected, it was found that only a small subset of the PBMCs werestained. These results demonstrate that LtxA binds to specific WBCs andthat LtxA targets only cells with activated surface LFA-1.

Example 2

It was known that the initial steps leading to inflammation include thebinding of activated PBMCs to endothelial cells and migration across theendothelial barrier. Strategies to treat inflammatory disorderstherefore include depletion of those WBCs that are involved inpathogenesis and interruption of their binding and migration into theaffected tissue. In this example, assays were carried out to determineif LtxA could block binding of activated PBMCs to human brainendothelial cells (HBECs) to model the initial steps leading toinflammation.

More specifically, D3 or 5i HBECs were grown to monolayer on collagen orgelatin and then stimulated with TNF. Calcein-labeled PBMCs activatedwith PMA were then added to the monolayer and incubated for 2 hours.Unbound cells were washed and the percent PBMC binding was calculated bymeasuring fluorescence. It was found that LtxA was able to preventbinding of activated PBMCs to the endothelial cells in a dose-dependentmanner (FIG. 4). Greater than 50% blocking was observed at higher dosesof LtxA. Because these PBMCs were from a healthy individual, completeblocking was not observed since most of the cells were normal and notaffected by LtxA. Cytotoxicity assays showed that only approximately10-20% of the cells stained with propidium iodide after two hoursthereby indicating that many of the cells that were blocked by LtxA werenot killed. Thus, the above results demonstrate that LtxA has theability to selectively deplete and block activated PBMCs from binding toendothelial cells.

In addition, in an in vitro model for multiple sclerosis (MS), it wasfound that LtxA blocked the migration of monocytes across a HBEC layer(FIG. 5). This model represents migration of WBCs into the CNS, whichoccurs in MS. These results suggest that LtxA can be used in treating MSvia its ability to selectively deplete and/or block monocytes fromentering into the CNS.

Example 3

In this example, assays were carried out to compare the effects of LtxAand anti-LFA-1 mAb EFALIZUMAB on T-cells. More specifically, 5×10⁵Jurkat cells/ml were treated with serial dilutions of LtxA or EFALIZUMABfor 72 hours before the proliferation of the cells were examined usingthe CELLTITER GLO kit from PROMEGA. It was found that LtxA decreasedproliferation of the cells in a dose-dependent manner (FIG. 6). Theinhibition of proliferation increased over time with an IC₅₀ value of250 ng/ml after 72 hours. EFALIZUMAB also decreased proliferation of thecells in a dose-dependent manner; but the time of stimulation did notseem to augment this effect (FIG. 6). The IC₅₀ value for EFALIZUMAB was5 mg/ml at all times. Thus, the effective dose of LtxA is approximately20,000 times lower than that of EFALIZUMAB.

Assays were also carried out to examine the efficacy of LtxA andEFALIZUMAB on activated PBMCs from psoriasis patients. Briefly, PBMCswere isolated from ten donors with severe plaque psoriasis (threefemales and seven males, age 29-63 (48+13)). PBMCs were isolated fromblood samples by centrifugation over a lymphoprep density gradient andimmediately frozen in liquid nitrogen until used. The cells were thenactivated using Staphylococcal enterotoxin B (SEB) at a finalconcentration of 1 μg/ml and treated with dilutions of LtxA orEFALIZUMAB. It was found that LtxA inhibited proliferation of theactivated PBMCs from psoriasis patients and the effective doses were atleast 5000 times lower than those of EFALIZUMAB (FIG. 7).

Example 4

In this example, assays were carried out to evaluate the effect of LtxAand EFALIZUMAB in alleviating psoriasis in vivo in a psoriasis xenografttransplantation model. As mentioned above, LFA-1 is a heterodimerconsisting of CD11a (αL) and CD18 (132) integrin subunits. It binds tointercellular adhesion molecule 1 (ICAM-1) present on antigen-presentingcells (APCs) and functions as an adhesion molecule. Binding betweenLFA-1 and ICAM-1 results in induction of T-cell activation, but alsoallows anchoring of these cells to the endothelium which is followed byextravasation leading to recruitment of T-cells to the site ofinflammation.

Methods Patient Material:

Two patients with plaques-type psoriasis were identified and keratomeskin biopsies from lesional skin were obtained. The patient's psoriasiswas un-treated for at least 1 month prior to the time of skin removal.

Xenograft Transplantation Protocol:

Keratome skin biopsies were cut into smaller pieces beforetransplantation on the back of anesthetized SCID mice (female, 6-8 weekof age, M&B Taconic, Denmark). After a healing period of approximately10 days, the animals were divided into separate treatment groups. Atotal of 24 mice were entered into the study. Animals were allocated totwo consecutive study series, each representing skin grafts from oneindividual psoriasis patient (total of 2 patients, 12 mice for eachseries). Each series was subdivided into 4 groups. One group in eachseries served as untreated control whereas the other groups wereallocated to treatment with LtxA, EFALIZUMAB, or LtxA vehicle. Theallocation scheme was as follows:

Mice pr group Total mice Group per series Treatment per treatment 1 2untreated 4 2 4 EFALIZUMAB 8 3 4 LtxA 8 4 2 LtxA vehicle 4

LtxA (0.5 mg/kg), EFALIZUMAB (6 mg/kg), and LtxA vehicle (Trisbuffer/NaCl) was administered once daily by i.p. injection for 3 weeks.These dosages were chosen as it was demonstrated that 2 mg/kg LtxA had anon-toxic therapeutic effect when it was administered in a mouseleukemia xenograft model (Kachlany, S. C. et al., Leukemia Research2010; 34:777-85). Similarly, administration of 6 mg/kg EFALIZUMAB (i.p.daily) or 10 mg/kg anti-ICAM-1 (i.p. every second day) to mice in thepsoriasis xenograft transplantation model demonstrated a therapeuticeffect (Zeigler M et. al., Lab Invest 2001; 81: 1253-61 and Boehncke W Het. al., Br J Dermatol 2005; 153: 758-66).

During treatment, human psoriatic skin grafts were clinically assessedtwice weekly and given a semi-quantitative clinical psoriasis scoreaccording to the clinical signs: scaliness, induration, and erythema.The parameters were scored using the three-point scale: 0=complete lackof cutaneous involvement; 1=slight involvement; 2=moderate involvement;3=severe involvement. On this scale from 0 to 3, a maximal score of 3represents severe scale, induration, and erythema of the psoriaticxenografts.

After 3 weeks and after the final clinical assessment, the animals werekilled and 4-mm size punch biopsies were taken centrally from each humanpsoriatic skin graft. The biopsy samples were paraffin embedded andstained with haematoxylin/eosin (HE), On five HE stained sections thefollowing parameters were assessed: 1) epidermal thickness, 2)parakeratosis, 3) psoriasis pattern, 4) angiogenesis, 5) lymphocytes,and 6) stratum granulosum. Epidermal thickness was measured as thedistance from stratum corneum to the deepest part of the rete pegs.Parakeratosis, psoriasis pattern, angiogenesis and lymphocytes wereevaluated and given scores in the range 0-4 where 0 denotes no psoriasisand 4 denotes full fledge psoriasis. Stratum granulosum was evaluated inthe range 0-4 where 0 denotes full fledge psoriasis and 4 no psoriasis.

Statistic Analysis:

Results are shown as mean±SEM. The non-parametric Mann Whitney test wasused to test for differences between treatment groups insemiquantitative clinical psoriasis scores, parakeratosis scores,psoriasis pattern scores, angiogenesis scores, lymphocyte scores, andstratum granulosum scores. Students t-test was used to test for nodifferences between treatment groups for epidermal thickness.Observations made for different mice were assumed to be independent ofeach other. All tests were two-sided and p values <0.05 were consideredsignificant.

Results Control Treatment (Untreated Versus LtxA Vehicle):

No differences between the vehicle and the untreated groups were foundin the semi-quantitative clinical psoriasis, the parakeratosis, thepsoriasis pattern, the angiogenesis, the lymphocyte, the stratumgranulosum scores, and the epidermal thickness measures for bothpatients. Therefore these two groups are pooled in the following andnamed negative control group.

Also, it was found that the mice treated with LtxA did not show anyphysiological changes during the study period, and their bodyweightincreased throughout the study similarly to the negative control treatedmice.

Semiquantitative Clinical Psoriasis Score:

Semi-quantitative clinical psoriasis scores provide a superficialevaluation of the graft where the histological scores show a more indepth status. Reduction in epidermal thickness is considered the finalendpoint when evaluating the treatment effect.

Semiquantitative clinical psoriasis scores were obtained twice weeklythroughout the study. As shown in FIG. 8, LtxA significantly decreasedthe semiquantitative clinical psoriasis score (p<0.001) by 3 weekstreatment. EFALIZUMAB also decreased the semiquantitative clinicalpsoriasis score (p=0.094) by 3 weeks treatment, however not to asignificant degree.

Epidermal Thickness:

After 3 weeks treatment, the mice were killed and biopsies taken fromthe human psoriatic skin graft. Epidermal thickness was measured on HEstained paraffin embedded sections. It was found that LtxA (p=0.002) andEFALIZUMAB (p=0.024) significantly decreased the epidermal thickness.See FIG. 8.

Psoriasis Pattern Scores:

Psoriasis pattern scores give an overall assessment of the psoriaticphenotype observed in the HE stained sections and sums up the results ofthe epidermal thickness measure, the parakeratosis, the angiogenesis,the lymphocyte, and the stratum granulosum scores. As shown in FIG. 9,LtxA significantly decreased the psoriasis pattern score (p=0.029) by 3weeks treatment. EFALIZUMAB also decreased the psoriasis pattern score(p=0.059) by 3 weeks treatment, however not to a significant degree.

Parakeratosis Scores:

The parakeratosis scores provide an assessment of the degree ofparakeratosis observed in epidermis. Due to the increased turnover anddecreased differentiation of keratinocytes in psoriatic skin,parakeratosis (presence of nucleus in desquamated cells) is oftenpresent in psoriatic skin. In this study, it was found that LtxA(p=0.282) and EFALIZUMAB (p=0.059) decreased the parakeratosis score by3 weeks treatment, however not to a significant degree. See FIG. 9.

Angiogenesis Scores:

Angiogenesis scores provide an assessment of the degree ofvascularization observed in the dermal compartment. Dermis of psoriaticskin is highly vascularized as compared to the dermis of healthy skin.In this study, it was found that LtxA (p=0.181) and EFALIZUMAB (p=0.081)decreased the angiogenesis score by 3 weeks treatment, however not to asignificant degree See FIG. 10.

Lymphocyte Scores:

Lymphocyte scores provide an assessment of the degree of lymphocyticinfiltrate present both in the dermal and the epidermal compartment.Psoriatic skin is characterized by an increased infiltrate oflymphocytes compared to healthy skin. In this study, it was found thatLtxA (p=0.005) and EFALIZUMAB (p=0.008) both significantly decreased thelymphocyte score by 3 weeks treatment. See FIG. 10.

Stratum Granulosum Scores:

Stratum granulosum scores provide an assessment of the degree of stratumgranulosum presence in the epidermis. Due to the increased turnover anddecreased differentiation of keratinocytes in psoriatic skin, this celllayer is typically diminished or lost in psoriatic skin. In this study,it was found that LtxA decreased the stratum granulosum score (p=0.081)by 3 weeks treatment, however not to a significant degree. EFALIZUMABsignificantly decreased the stratum granulosum score (p=0.012) by 3weeks treatment. See FIG. 11.

In sum, the above results demonstrated that the three-week treatmentwith LtxA significantly decreased the semi-quantitative clinicalpsoriasis score, the epidermal thickness, psoriasis pattern, and thelymphocyte scores. EFALIZUMAB significantly decreased the epidermalthickness and the lymphocyte scores. Also, this study demonstrated thatLtxA significantly alleviated the psoriatic phenotype in the graftedskin from both patients. EFALIZUMAB significantly alleviated theepidermal thickness but not the clinical phenotype of psoriasis. Thus,the results suggest that LtxA is more effective than EFALIZUMAB intreating psoriasis.

Example 5

In this example, general materials and methods for carrying out variousassays are disclosed regarding LtxA and HIV.

Cells:

PBMCs from healthy donors were isolated from leukopacs (New York BloodCenter), whereas PBMCs from HIV-infected subjects were from whole blood.The use of human specimens for this study was approved by aninstitutional review board. After Ficoll-Paque Plus centrifugation,PBMCs were used directly in the experiments or were first enriched forCD4 T cells using a negative-selection magnetic bead kit (MILTENYIBIOTECH or INVITROGEN) as described in Vasiliver-Shamis et al. 2009. JVirol 83:11341-11355.

Leukotoxin (LtxA):

LtxA was purified from culture supernatants of A. actinomycetemcomitansstrain NJ4500 as described in Diaz R., et al. 2006. Microb Pathog40:48-55 and Kachlany, S. C., et al. 2002. Protein Expr Purif 25:465-71.Protein was lyophilized in sterile vials and stored at −80° C. Sampleswere reconstituted in sterile distilled water and filtered through a0.22 μm filter prior to use. When prepared in this manner, LtxA wasstable for at least 6 months.

Planar Bilayer Assay and Microscopy:

Planar bilayers were prepared from liposomes containing 12.5%Ni²⁺-chelating DOGS-NTA(1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl)iminodiaceticacid] succinyl and glycosylphosphatidylinositol (GPI)-anchoredCy5-labeled mouse ICAM-1 (density of 200-250 molecules/μm²) as describedin Vasiliver-Shamis et al. 2009. J Virol 83:11341-11355 andVasiliver-Shamis et al. 2008. J Virol 82:9445-9457. His₆ gp120 of HIV-1DH12 used to reconstitute the bilayers was produced from recombinantvaccinia virus (Cho, M. W., et al. 2001. J Virol 75:2224-2234), labeledwith ALEXA FLUOR 488 (INVITROGEN), and applied onto the bilayers at aconcentration that resulted in gp120 density of 200 to 250molecules/μm². After the flow cell containing the bilayers was warmed to37° C., cells were injected and images collected for 1 hr on awide-field fluorescence microscope. To test the effects of anti-gp120mAbs, bilayers were first treated for 20 min with 20 μg/ml of each mAb(EH21, 2219, and 654). The cells were also suspended in a buffercontaining 20 μg/ml of the mAb before injection to the bilayers.

Multicolor fluorescence microscopy was performed on an automatedmicroscope with an Orca-ER cooled charge-couple-devise (CCD) camera orelectron multiplier CCD camera (HAMAMATSU). The hardware on themicroscope was controlled using SCANALYTICS IP-LAB software on a DELLpersonal computer. Image processing and analyses were performed with IPLAB and METAMORPH software.

Flow Cytometric Analysis:

Surface and intracellular staining of PBMCs was done as described inKaur, et al. 2007. Virology 369:214-225. Fluorescence-conjugatedantibodies to CD3 (APC-Cy7) and CD8 (APC) were used to gate CD4 T cellpopulation (CD3+CD8-) studied, and FITC-conjugated anti-p24 monoclonalantibody (KC57; COULTER) was used to detect CD4 T cells with active HIVreplication. LFA-1 expression was measured with PE-conjugated anti-CD11amAb (BD PHARMINGEN). Data analyses were done with the FLOWJO software(TREE STAR).

Cell Viability Assay:

CD4 T cells (1×10⁵/well) were added to microtiter wells pre-coated withwild type, mutant, or no gp120, or with soluble gp120, and then treatedwith LtxA at designated concentrations. For control, cells activatedwith anti-CD3/anti-CD28 on microtiter wells were also treated with LtxA.After 20 hrs, cellular viability after LtxA treatment was determinedusing the CELLTITER-GLO luminescent cell viability assay (PROMEGA).Plates were read in PERKIN ELMER VICTOR3 Multilabel Counter in theluminescence mode.

Real-Time PCR:

Cells were lysed by incubation with a lysis buffer (5 mM Tris [pH 8.3],0.45% TRITON X-100, 0.45% TWEEN 20) and proteinase K (20 mg/ml) for 1 hrat 60° C. and then for 15 min at 95° C. to inactivate proteinase K. Celllysate (2 μl each) was then used in a 20 μl reaction on the APPLIEDBIOSYSTEMS 7900HT FAST REAL-TIME PCR System with 1×SYBR Green SUPERMIX(SYBR Green I Dye, AMPLITAQ GOLD® DNA Polymerase, dNTPs with dUTP,passive reference dye, and optimized buffer components) (APPLIEDBIOSYSTEMS) and the specific primers (5 picomoles each). For measuringgag, a 115-bp fragment in the gag region was amplified using the primersSK38 (5′-ATAATCCACCTATCCCAGTAGGAGAAAT-3′, SEQ ID NO: 3) and SK39(5′-TTTGGTCCTTGTCTTATGTCCAGAATGC-3 SEQ ID NO: 4) (INVITROGEN) under thefollowing reaction condition: 95° C. for 10 min (initial denaturation)and 40 cycles of 95° C. for 15 sec (denaturation) and 60° C. for 1 min(primer annealing and extension). For β-actin, a 217 bp fragment wasamplified using primers 5′-CTCCATCCTGGCCTCGCTGT-3′ and5′-CACCTTCACCGTTCCAGTTT-3′ (SEQ ID NOs: 5 and 6) in the followingreaction condition: 95° C. for 10 min (initial denaturation) and 40cycles of 95° C. for 30 sec (denaturation), 55° C. for 30 sec (primerannealing), 60° C. for 1 min (primer extension). PCR products werequantified based on the standard curve in each experiment. The 8E5 LAVcells, each of which contains 1 copy of HIV provirus and 2 copies ofβ-actin gene, were used to generate the standard curves.

Example 6

In this example the specificity of LtxA for activated PMBCs was examinedMore specifically, malignant human monocyte cell line, THP-1, and PBMCsfrom four healthy adults were treated with LtxA at differentconcentrations for 24 hours. Cell viability was determined bymeasurement of cellular ATP. The results are shown in FIG. 12A, whereuntreated samples represent a relative viability of 1.0. The curve forPBMCs represents the average of the four human PBMC samples performed inquadruplicate. As shown in the figure, the cells were generallyresistant to killing by LtxA compared to the sensitive leukemia cellline, THP-1 (FIG. 12A).

One hypothesis why some cells are more sensitive to LtxA than others isthat LtxA recognizes the activated form of LFA-1 better than LFA-1 inthe resting state. To test this, an assay was carried out using JurkatT-cell line that expresses a high level of constitutively active LFA-1(J-β_(2.7)/LFA-1Δ). As controls, isogenic cell lines that either expressthe wild type form of LFA-1 (J-β_(2.7)/LFA-1 wt) or lack LFA-1expression completely (J-β_(2.7)/mock) were used. It was found thatcells with activated LFA-1 were ten times more sensitive toLtxA-mediated toxicity than cells with resting state LFA-1 andLFA-1-deficient cells were not affected by the toxin (FIG. 12B). Thus,LtxA is more toxic towards WBCs expressing activated form of LFA-1,which are the same type of cells that are preferentially infected byHIV.

The data presented above using the Jurkat cell lines demonstrates thatLtxA prefers the activated form of LFA-1 as a target. Furthermore, basedon results showing that only a small fraction of healthy PBMCs areaffected by LtxA, it is possible that the killed cells represent thesmall fraction of activated cells in the population. To test thishypothesis, assays were carried out to determine if LtxA retained thisspecificity for activated LFA-1 in a sample of PBMCs from healthyindividuals. After treatment of PBMCs with LtxA, cells were stained forCD3, CD4, and LFA-1, fixed, and analyzed by flow cytometry. It was foundthat approximately 11% of the CD3+T lymphocyte population expressed highlevels of LFA-1 while 78% expressed low levels (the remaining did notexpress LFA-1) prior to treatment with LtxA (FIG. 13). Following LtxAtreatment, the high LFA-1-expressing cells were almost completelyeliminated (a decrease to 0.4%) while the lymphocytes expressinglow-levels of LFA-1 (or not expressing LFA-1) were essentiallyunaffected. The LFA-1^(hi+) cell population consisted of 96% CD3⁺ CD4⁺T-cells. These results show that LtxA is able to selectively killactivated CD4⁺ T-lymphocytes in a mixture of PBMCs, suggesting thatnewly HIV-exposed or infected T-cells would be desired targets for LtxA.

Example 7

In this example, assays were carried out to show effects of LtxA inHIV-infected T-cells. To determine if LtxA could target LFA-1⁺ T-cellsthat were infected with HIV, HIV-GFP was used to infect T-cells witheither high levels of LFA-1 or not expressing LFA-1. The samples werethen treated with LtxA to examine the population that was depleted.Briefly, T-cells bearing LFA-1 and HIV-GFP were mixed with T cellsbearing no LFA-1 and then treated with LtxA. The flow cytometry datashow that LtxA was able to deplete LFA-1⁺ cells infected with HIV, butdid not affect the population of LFA-1⁻ cells with no virus (FIG. 14).Thus, the results demonstrate LtxA has the capacity to specificallytarget LFA-1⁺ cells that harbor HIV.

Assays were also carried out to show effects of LtxA on HIV-PBMCsnaturally infected with HIV-1 from an HIV-positive patient with a highviral load. Specifically, PBMCs from an HIV-positive individual weretreated with LtxA for 24 hours and then intracellular viral DNAremaining was measured by PCR. Viral DNA (not RNA) which identifiesintracellular virus (including latent infection) was assayed. AnHIV-negative individual was included as a control. It was found that asignificant drop in viral DNA occurred after a 24-hour treatment of LtxA(FIG. 15). Thus, the results demonstrate that LtxA is able to depletelatently-infected cells from an HIV-positive patient.

Example 8

In this example, assays were carried out to show that the interaction ofCD4 T cells with HIV-1 gp120 on bilayers triggers LFA-1 activation andsupramolecular re-organization.

Briefly, resting native CD4 T cells were obtained ex vivo from theperipheral blood of healthy HIV-seronegative donors. The interaction ofthe cells with gp120 and ICAM-1 was analyzed on glass-supported planarlipid bilayers. The bilayers served as an experimental model mimickingthe virion surface or the infected cell surface (Vasiliver-Shamis, etal. 2009. J Virol 83:11341-11355). To discern changes in cellularmorphology and molecular organization, the bilayers were loaded withALEXA FLUOR 488-labeled gp120 and Cy5-labeled ICAM-1. For comparison,bilayers were also prepared with Cy5-labeled ICAM-1 alone. After thecells were added onto the bilayers, live images were acquired for up to1 hr using multicolor fluorescence microscopy.

Native peripheral CD4 T cells express LFA-1 in the inactive states withlow affinity for ICAM-1. Therefore, these cells rarely form contact withbilayers containing ICAM-1 alone and if they do, the contact istransient.

As shown in FIG. 20, native CD4 T cells did not form stable interactionwith the ICAM-1 bilayer. The native CD4 T cells were injected onto abilayer containing only ICAM-1 and monitored over one hour for thepresence of cells (FIG. 20, bright-field panels), contact with thebilayer (interference reflection microscopy (IRM) panels) and contactwith ICAM-1 (ICAM-1 panel). The same representative field is shown atthe indicated time points. Thin black arrows show the cells thatinteracted transiently with the bilayers at 15 min but had no ICAM-1accumulation and migrated by 30 min. Three new cells interacting withthe bilayer at 50 min were also observed and indicated by thick whitearrows; again these cells made no ICAM-1 contact.

However, when these cells were introduced onto bilayers containing gp120and ICAM-1, cells established gp120 contacts within the first 5-10 minand 10 min later started forming ICAM-1 contact (FIG. 16A). About 40% ofthe cells established stable LFA-1-ICAM-1-mediated adhesion (FIG. 16B).In addition, supramolecular rearrangements of both gp120 and ICAM-1 wereobserved: gp120 accumulated into a central cluster and ICAM-1 assembledinto a ring, which is either symmetrical (for 20% of the cells; FIG.16B, top) or asymmetrical (for another 20%; FIG. 16B, bottom), aroundthe central gp120 cluster.

Once formed, this morphology was maintained for the duration of theexperiment (1 hr). The LFA-1-ICAM-1 interaction and rearrangement weretriggered specifically by gp120 binding to its receptors on the T cellsurface, since pre-treatment with mAbs (20 μg/ml) to the CD4-bindingsite (654) substantially reduced not only the numbers of cells forminggp120 contact (FIG. 16C, left graph), but also ICAM-1 contact (FIG. 16C,right graph). A mAb (20 μg/ml) against the V3 loop (2219), which isinvolved in binding the chemokine receptor, also decreased both gp120and ICAM-1 contacts, while a control mAb to the N-terminus of gp120(EH21) which does not participate in CD4 or the chemokine receptorbinding had no effect.

The above results demonstrate that gp120 binding to CD4 and theco-receptor on fully quiescent CD4 T cells triggers LFA-1 activation andsupramolecular organization.

Example 9

As shown in EXAMPLE 8 above, LFA-1 activation is triggered by gp120 uponbinding to CD4 T cells. It is surmised that these cells should becomehighly susceptible to LtxA, a bacterial leukotoxin that is known topreferentially kill leukocytes with high levels of the activated form ofLFA-1. In this example, assays were carried out to show that CD4 T cellsexposed to surface-bound gp120 are more susceptible to killing by LtxA.

Specifically, resting CD4 T cells from healthy uninfected donors wereincubated on tissue culture wells coated with or without gp120 ((10μg/ml)) and then treated with different concentrations of LtxA. After 20hrs, cell viability was determined by measurement of cellular ATP. Amutant gp120 protein lacking the ability to bind CD4 and the chemokinereceptors (CD4bs− V3−) was tested as a control.

As shown in FIG. 17, CD4 T cells interacting with gp120 on the wellswere more susceptible to LtxA than the cells incubated with the mutatedgp120 or no gp120. The increased killing was apparent at differentconcentrations of LtxA. The 50% effective dose (ED50) of LtxA againstgp120-treated CD4 T cells was 367 ng/ml, while the ED50 of LtxA againstuntreated cells and mutant gp120-treated cells were 437 and 440 ng/ml,respectively.

Furthermore, R5 gp120 (JRFL) and X4 gp120 (HXB2) was tested forcomparison. CD4 T cells were treated with soluble gp120 oranti-CD3/anti-CD28 coated on microtiter wells prior to addition of LtxA.After incubation with LtxA for 20 hrs, the cell viability was measuredbased on cellular ATP. It was found that both R5 gp120 and X4 gp120mediated enhanced susceptibility to LtxA (FIG. 17). However, thisactivity was induced only when the CD4 T cells interacted with gp120bound on the well surface; soluble gp120 did not have the same effect(FIG. 17), indicating that gp120-mediated crosslinking of CD4 and/or thechemokine receptor is essential for LFA-1 activation that renders theCD4 T cells more susceptible to killing by LFA-1-specific LtxA. Forcomparison, CD4 T cells activated by surface-bound anti-CD3 andanti-CD28 antibodies were also treated with LtxA, and nearly 100% ofthese cells were killed by LtxA at the concentrations tested (390 and780 ng/ml) (FIG. 17). This contrasts to only ˜50% of untreated CD4 Tcells susceptible to LtxA at the same concentrations, confirming strongpreference of LtxA for LFA-1 on activated T cells.

The above results demonstrate that gp120 binding to quiescent CD4 Tcells renders the cells more susceptible to LtxA due to LFA-1 activationas a result of CD4 and/or the chemokine receptor crosslinking, but thegp120-induced activity is not as potent as that triggered by TCRengagement.

Example 10

In this example, assays were carried out to show that viralp24-producing CD4 T cells in the peripheral blood of HIV-infectedsubjects display higher levels of surface LFA-1 expression.

To evaluate LFA-1 expression on HIV-infected CD4 T cells in theperipheral blood, assay was conducted to analyze ex vivo PBMCs from twoviremic untreated HIV-infected subjects (PS05 and PS07) who wereasymptomatic and had CD4 count of >450. The PBMCs were stained with mAbsfor surface expression of CD3, CD8, and LFA-1, as well as forintracellular p24 antigen after permeabilization. The cells weresubjected to flow cytometric analyses, and the data analyzed by theFlowJo software. The results are shown in FIG. 18, where the dot plots(top panels) show that 0.57% and 0.52% of p24+CD3+CD8− T cells weredetected in subjects PS05 and PS07, respectively. The p24+ gating isbased on the p24 staining of HIV-seronegative PBMCs tested in parallelin each assay. See FIG. 21A, which shows the background p24 staining ofCD4 T cells (CD3+CD8-) from a HIV-seronegative donor, NG05. This gatingwas used to determine positive p24 staining in the CD4 T cells ofHIV-seropositive subjects.

When the LFA-1 expression of p24+ and p24− cells was compared, highermean fluorescence intensity (mfi) levels were detected on the p24+ cellpopulation than on the p24− cell population from the same subjects,indicative of the activated state of HIV-infected CD4 T cells producingviral p24 antigens in the peripheral blood (FIG. 18). The histograms(bottom) compared LFA-1 expression on p24+ and p24− CD4 T cells; the mfifor p24+ and p24− CD4 T cells are 462 and 376 for PS05, and 311 and 228for PS07. The higher LFA-1 expression levels were specific as noincrease in CD3 expression was observed on p24+ cells as compared top24− cells (FIG. 21B). Similarly, as shown in FIG. 21B, CD3 expressionwas found on p24+ and p24− CD4 T cell populations from HIV-infectedsubjects PS05 and PS07. The mfi for p24+ and p24− cells are 87 and 85for PS05, and 74 and 61 for PS07.

Example 11

CD4 T cells supporting active HIV infection have higher levels ofsurface LFA-1 expression and LtxA has been shown to preferentiallytarget activated CD4 T cells expressing higher levels of LFA-1, it wasexamined whether LtxA treatment can target the infected CD4 T cells andreduce the levels of viral DNA in the PBMCs of HIV-infected individuals.

PBMCs from two viremic HIV-infected subjects (PS05 with 38,165 vRNAcopies/ml and CD4 count of 814 and PS14 with 21,815 vRNA copies/ml andCD4 count of 494) were treated with LtxA (7.8 μg/ml) for 20 hrs. Theviral DNA and β-actin DNA were quantified by real time PCR with thespecific primers. See FIG. 19A. Averages and standard deviation from 4-5repeat experiments are presented.

As shown in FIG. 19A, 2682 and 1223 copies of viral DNA were initiallydetected in PBMCs of subjects PS05 (˜2.5×10⁵ cells) and PS014 (˜1.2×10⁵cells), respectively, and LtxA treatment reduced the amounts of viralDNA by 84% and 39%. The reduction of viral DNA was accompaniedrespectively by 91% and 68% loss of PBMCs as indicated by β-actinmeasurement (FIG. 19B). The higher levels of cell loss relative to viralDNA reduction are not surprising, since not all cells sensitive to LtxAkilling are infected by the virus. Indeed, HIV infection has beenconsistently associated with increased numbers of activated uninfectedbystander CD4 and CD8 T lymphocytes (Giorgi, et al. 1993. J AcquirImmune Defic Syndr 6:904-912, and Hazenberg, et al. 2003. Aids17:1881-1888).

In sum, the above study demonstrates that HIV-1 itself is capable ofstimulating LFA-1 and converting it from an inactive state to an activeconformation that allows a high affinity binding for the ICAM-1 ligand.It also discloses that LtxA is effective at removing cells bearing HIV-1and reducing viral DNA loads due to its cytotoxic activity againstactivated LFA-1^(hi+) cells that are most efficient to supportproductive HIV infection. These results were surprising since, unlikeCD4 and the chemokine receptors, LFA-1 and ICAM-1 are not required forHIV infection.

The foregoing example and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims.

What is claimed is:
 1. A method for treating Crohn's disease, consistingessentially of administering to a subject in need thereof an effectiveamount of a leukotoxin comprising SEQ ID NO: 1, wherein the Crohn'sdisease is characterized by an influx of immune cells into theintestines and activation of leukocytes that express upregulated andactivated leukocyte function antigen (LFA-1) on their surface to whichthe leukotoxin specifically binds.
 2. The method of claim 1, wherein theleukotoxin is prepared from Aggregatibacter actinomycetemcomitans. 3.The method of claim 1, wherein the leukotoxin is purified.
 4. The methodof claim 1, wherein the leukotoxin is administered with apharmaceutically acceptable carrier.